Proteinous material



Patented Jan. 9, 1945 UNITED PROTEINOUS Arnold K. Balls, Washington, D.0., Walter S. Hale, Alexandria, Va., and Thomas E. Harris, Washington,D. 0., asslgnors to Claude R. Wickard, as Secretary of Agriculture ofthe- United States of America, and his successors in oillce No Drawing.Application February 8, 1943, n Serial No. 475,170

9 Claims. (01. sec-112) (Granted under the 'act of March 3. 1883. asamended April so, 1928; 370 o. a. 757) 'tion' products. The sulfurcontent is among the This application is made under the act of.

March 3, 1883, as amended by the act of April 30, 1928, and theinventionherein described, if patented may. be manufactured and used byor for the Government of the United States of America forgovernmental-purposes without the payment to us of any royalty thereon.

This invention relates to proteinous materials, and more particularly toproteinous materials derived from vegetable products.

We havefound that a new sulfur containing proteinous material, which ishighly toxic to cervents. Suitable fat solvents are petroleum ether andother low boiling petroleum fractions, ethyl ether, benzene,*toluene,chlorinated hydrocarbon solvents and the like. The extraction may becarried out by lixiviation according to the process described in theapplication for patent, Serial No. 343,564, filed July 2, 1940. p

Hydrolysis of the sulfhydryl compounds can be effected by means of astrong mineral acid, such as sulfuric or hydrochloric acid, preferablyin the presence of a suitable solvent, such as ethyl ether. From thehydrolyzate, which comprises a number of substances of undeterminedconstitution, the hydrochloric acid salt of the new sulfur-containingproteinous material can be isolated by fractional precipitation fromaqueous ethyl alcohol, and this salt canbe obtained in substantiallypure crystalline form.

The new proteinous material is precipitatedupon addition of ethylalcohol to a neutralized solution of the hydrochloride in water. Thehydrochloride appears to be the salt of a substance related toprotamines. It yields amino acids on hydrolysis and is incompletelyprecipitated by sulfosalicylic:and by trichlcroacetic acid. Measurementsof the rate of diffusion indicate a value of about 10,000, but notexceeding 12,000, for the molecular weight of the hydrochloride whileanalytical data show that its molecular weight must be at least 6,000.The'new proteinous material possesses properties of a compound lying onthe borderline between proteins and their degradahighest known forproteins. This sulfur apears as cystine and may be reversiblyoxidized-and reduced.

Production of the hydrochloride of the proteinous material from wheatflour is illustrated by the following example:

Using a percolator, 200 pounds of wheat flour was extracted at roomtemperature with 30 gallons of petroleum ether (boiling range .30- 60C.) for eight hours. Practically all of the solvent was removed from theextract by distillation in vacuum and the dark-colored, oily residue waskept at about -1.5" C. for several weeks, after which it was centrifugedunder refrigeration to remove the precipitate of sterol crystals whichhad deposited during the cold storage. The thick, oily substance thusobtained was dissolved in about an equal volume of ethyl ether and theether solution was treated with about one and one-half its volume ofcold 1 N-hydrochloric acid in absolute ethyl alcohol andjallowed tostand for one hour at about 0 C. The hydrolyzate that formed wasseparated in a centrifuge and washed first with absolute alcohol andthen with ethyl ether. V

The hydrolyzate was then dissolved in mi. of 'water and the solution wasmixed with 300 ml. of absolute ethyl alcohol. A precipitate formed andwas removed in a centrifuge. .The clear liquid remaining wasconcentrated on a steam bath and then evaporated to dryness in vacuum.There was obtained 16.8 grams of the crude hydrochloride of. thesulfur-containing proteinous material.

The crude hydrochloride in the amount of 15.5 grams was dissolved in 25ml. of water, 225 ml. of absolute ethyl alcohol wasadded, and thesolution was allowed to standat 5 Q'for four hours. A crystallineprecipitate of the hydrochloride was formed weighing 4.10 grams.

Using the same procedure the hydrochloride was recrystallized a secondand a third time. The following results were obtained from analysisafter each crystallization:

Nitrogen Chlorine Sulfur Per cent Per cent Per cent crystallized once16. 20 6. 64 4. 40 Crystallized twice. 33 g 46 Crystallized three times{g The hydrochloride is very soluble inwater and The results of ananalysis of the thrice-crys-,

tallized material are given in the following table:

Percent by weight Chlorine (Pregl) 6.57 Sulfur (Pregl) 4.42

Nitrogen (Kjeldahl after Pregl) 17.35 Amino nitrogen (Van Slyke) 1.65Arginine (Thomas, Ingalls 8; Luck) 20.4 Cystine (Sullivan 8: Hess) 15,7Tyrosine (Bernhard) 3.0

Having thus described our invention, we claim:

1. A hydrochloric acid salt of a proteinous material derived fromsulfhydryl compound isolated from a cereal grain, said salt beingcrysstallizable and containing approximately 17 percent nitrogen and 4.5percent sulfur by weight, yielding on hydrolysis at least 50 percent ofthe nitrogen as amino acids of the group consisting of arginine, cystineand tyrosine, and having a molecular weight not less than 6,000 and notexceeding 12,000.

2. The produce defined in claim 1 further characterized in that thecereal grain is wheat.

3. A crystalline hydrochloride of a proteinous material derived fromsulfhydryl compounds isolated from a cereal grain, said hydrochloridecontaining approximately 17 percent nitrogen and 4.5 percent sulfur byweight, yielding on hydrolysis at least 50 percent of the nitrogen asamino acids of the group consisting of arginine, cystine and tyrosine,and'having a molecular weight not less than 6,000 and not exceeding12,000.

4. The product defined in claim 3 further characterized in that thecereal grain is wheat.

5. The process which comprises hydrolyzing sulfhydryl compounds derivedfrom a cereal grain in hydrochloric acid, thereby obtaining thehydrochloric acid salt of a sulfur-containing proteinous material as aconstituent of the hydrolyzate, dissolvin the hydrolyzate in Water,fractionally precipitating the resulting solution by mixing suchsolution with absolute ethyl alcohol, evaporating the remaining liquidto dryness, dissolving the resulting material in water and thenprecipitating said hydrochloric acid salt from aqueous solution byadding absolute ethyl alcohol, whereby said hydrochloric acid salt isobsalt is obtained in substantially pure crystallinev i'orm, containingapproximately 17 percent nitrogen and 4.5 percent sulfur by weight,yielding on hydrolysis at least 50 percent of the nitrogen as aminoacids of the group consisting of arginine, cystine and tyrosine, andhaving a molecular weight not less than 6,000 and not exceeding 12,000.

'7. The process which comprises hydrolyzing a solution in ethyl ether ofsulfhydryl compounds derived from wheat with a solution of lN-hydrochloric acid in absolute ethyl alcohol, thereby obtaining thehydrochloric acid salt of a sulfurcontaining proteinous material as aconstituent of the hydrolyzate, dissolving the hydrolyzate in water,fractionally precipitating th resulting solution by mixing such solutionwith absolute ethyl alcohol, evaporating the remaining liquid todryness, dissolving the resulting material in water and thenprecipitating said hydrochloric acid salt from aqueous solution byadding absolute ethyl alcohol, whereby said hydrochloric acid salt isobtained in substantially pure crystalline form, containingapproximately 17 percent nitrogen and 4.5 percent sulfur by weight,yielding on hydrolysis at least 50 percent of the nitrogen as aminoacids of the group consisting of arginine, cystine and tyrosine, andhaving a molecular weiglat not less than 6,000 and not exceeding 12,00

8. The process which comprises hydrolyzing sulfhydryl compounds derivedfrom a cereal grain in hydrochloric acid, thereby obtaining thehydrochloric acid salt of a sulfur-containing proteinous material as aconstituent. of the hydrolyzate, dissolving the hydrolyzate in water,fractionally' precipitating the resulting solution by mixing suchsolution with absolute ethyl al cohol, evaporating the liquid remainingfrom said fractional precipitation to dryness, dissolving the resultingmaterial in water and then adding absolute ethyl alcohol to the aqueoussolution of the material thus obtained, whereby said hydrochloric acidsalt is precipitated in substantially pure crystalline form.

9. The process of preparing a crystalline hydrochloride of a proteinousmaterial related to protamines which comprises hydrolyzing sulfmainingfrom said fractional precipitation to dryness, dissolving the resultingmaterial in water and then adding absolute ethyl alcohol to the aqueoussolution of the material thus obtained, whereby said hydrochloric acidsalt is precipitated in substantially pure crystalline form.

ARNOLD K. BALLS. WALTER S. HALE. THOMAS H. HARRIS.

